(1) Field of the Invention
The present invention relates to a stable bilayer or multilayer membrane with channels through the membrane and containing a phospholipid and a mixture of a polyhydroxybutyrate (PHB) and a polyphosphate. In particular, the present invention relates to a method for transporting an ion or molecule through the channel in the membrane. Further, the present invention relates to a method for assaying for compounds which block the channel. Finally, the present invention relates to a method for incorporating the channels in membranes.
(2) Description of Related Art
R-poly-3-hydroxybutyrates and inorganic polyphosphates (polyPi) are ancient and ubiquitous homopolymers whose biological roles are not well understood. PHB, a head-to-tail polymer of R-3-hydroxybutyrate, is best known as a high molecular weight (60,000 to 1,000,000) polymer deposited within inclusion bodies in many prokaryotes. PolyPi are linear chains of orthophosphate joined by phospho-anhydride bonds, which have a free energy of hydrolysis comparable to that of ATP. Reusch et al (R. N. Reusch and H. L. Sadoff. J. Bacteriol. 156, 778-788 (1983); R. N. Reusch, et al., J. Bacteriol. 168, 553-562 (1986); and R. N. Reusch. Soc. Exp. Biol. and Med. 19, 377-381 (1989)) isolated PHB having a lower molecular weight (&lt;12,000) from bacterial plasma membranes, and from membranes and organelles of plants, and concluded that membrane PHB in Escherichia coli, Azotobacter vinelandii and Bacillus subtilis was complexed with Ca(polyPi). The presence of these complexes in bacterial membranes is discerned by observing the thermotropic fluorescence of the membrane probe, N-phenyl-1-naphthylamine; dissociation of the complexes gives rise to an increase in fluorescence with a peak at ca 56.degree. C. The concentration of PHB/polyPi is low during log-phase growth, but increases fifty to a hundred fold or more when the cells are made genetically competent whether by physiological or physico-chemical means. At high concentrations, the complexes cause alterations of the plasma membrane structure observable by freeze-fracture electron microscopy (R. Reusch, et al., Can. J. Microbiol. 33, 435-444 (1987)).
Poly-.beta.-hydroxybutyrate (PHB) and calcium polyphosphate complex membranes were extracted as biological complexes from bacterial membranes (Reusch, R., and Sadoff, H., Proc. National Acad. Science 85, 4176-4180 (1988)). Attempts to reconstitute the complex membranes from calcium polyphosphate and PHB in liposomes met with limited success, since they were significantly disassociated as can be seen from FIG. 1 of this reference. The putative functions of the biological complexes are further discussed in FEMS Microbiology Rev. 103, 119-130 (1992).
Reusch and Sadoff proposed a structure for the PHB/Ca(polyPi) in E. coli, based on molecular and computer modeling with regard for the physical properties of the polymers, the coordination geometry of calcium, and the membrane environment. It assumes that the amphophilic PHB forms a helical pore--with a lipophilic exterior of methyl and methylene groups and a hydrophilic lining of ester carbonyl oxygens--that is traversed by the more rigid polyPi anion. A channel is formed in the space between the two polymers which has solvating carbonyl oxygens column evenly spaced along its outer wall and negatively-charged binding sites at regular intervals along its inner wall. The channel is subdivided into several contiguous parallel lanes through which cations may move in single-file in the direction of concentration or voltage gradients. Since all cation binding sites are identical, the potential energy minima are also identical. This model of a multiple-site, single-file channel is consistent with current views on protein Ca.sup.2+ channel structure expressed by Hess and Tien and Almers and McCleskey (Hess, P. and R. W. Tsien. Nature. 309, 453 (1984); and Almers, W. and McCleskey, E. W., J. Physiol. 353, 585 (1984)). The model is used as a basis for explaining how the inventors believe the present invention functions; however, they do not want to be bound by any particular theory.